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Available online at www.sciencedirect.com Cell regulation by the Apc protein Apc as master regulator of epithelia Brooke M McCartney1 and Inke S Näthke2 The adenomatous polyposis coli (Apc) protein participates in many of the fundamental cellular processes that govern epithelial tissues: Apc is directly involved in regulating the availability of b-catenin for transcriptional de-repression of Tcf/ LEF transcription factors, it contributes to the stability of microtubules in interphase and mitosis, and has an impact on the dynamics of F-actin. Thus Apc contributes directly and/or indirectly to proliferation, differentiation, migration, and apoptosis. This particular multifunctionality can explain why disruption of Apc is especially detrimental for the epithelium of the gut, where Apc mutations are common in most cancers. We summarise recent data that shed light on the molecular mechanisms involved in the different functions of Apc. Addresses 1 Department of Biological Sciences, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA, USA 2 College of Life Sciences, Cell & Dev. Biology, University of Dundee, Dow Street, Dundee, Scotland, UK Corresponding author: Näthke, Inke S ([email protected]) Current Opinion in Cell Biology 2008, 20:186–193 This review comes from a themed issue on Cell regulation Edited by Alan Hall and Joan Massagué Available online 24th March 2008 0955-0674/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.ceb.2008.02.001 The initial identification of the adenomatous polyposis coli gene as the site of mutations in the familial form of colon cancer, familial adenomatous polyposis (FAP), was described in 1992 [1,2]. A causal relationship between Apc mutations and intestinal tract tumours was confirmed three years later with the establishment of the Min mouse model [3]. These mice are heterozygous for Apc and develop numerous intestinal tumours that mimic FAP. Subsequently, Apc has emerged as the most commonly mutated gene in colorectal cancer with reports of between 50 and >80% of sporadic tumours carrying such mutations. The search for how mutations in Apc initiate and/or support progression of tumours in the intestinal tract revealed that Apc is a multifunctional participant in a diverse array of cellular functions. In this review, we describe these diverse functions and their relationship to epithelial biology, and discuss recent significant Current Opinion in Cell Biology 2008, 20:186–193 advances in our understanding of their underlying molecular mechanisms. Wnt pathway regulation The first recognised function of Apc was its role in Wnt signalling [4,5]. This function is one of the driving forces for how mutations in Apc ensure that cells remain proliferative. Many of the molecular details of this pathway have been described extensively in many reviews [6]. Apc negatively regulates Wnt signalling by participating in the destruction complex, a complex that targets the key effector b-catenin for degradation (reviewed in [7]). However, the precise role of Apc in the destruction complex has not been clear. A number of recent papers employing largely biochemical and structural approaches have shed significant light on this question. The central region of the Apc protein contains three distinct types of repeats that are essential to its role in the negative regulation of Wnt signalling (Figure 1). Seven 20 amino acid repeats (20R) and three 15 amino acid repeats (15R) function in the binding and degradation of b-catenin, while three SAMP repeats bind to the RGS domain of Axin, a partner in the destruction complex (reviewed [7]). It has long been appreciated that phosphorylation of this central repeat region of APC significantly enhances its affinity for b-catenin [8]. The degree of conservation between repeats of the same type suggested that they might be functionally equivalent. However, recent work has clearly demonstrated that individual 15R and 20R differ significantly in their binding affinities for b-catenin [9]. This difference seems to be achieved in part by the N-terminal flanking regions of the repeats [9]. The 20R with the highest binding affinity to b-catenin (R3, 4 and 5) also contain a typical Tcf-like nine amino acid b-cateninbinding motif in their N-terminal flanking regions. The Tcf/LEF family of transcription factors partner with bcatenin in the nucleus to activate Wnt target genes. Changing a key aspartate residue in the flanking region to alanine resulted in a 30-fold reduction in the binding affinity for b-catenin. When the repeats are phosphorylated by the cooperative action of CK1 and GSK3b, the binding interaction is significantly altered and enhanced. Phosphorylation of any of the 20R, even those with no detectable binding to b-catenin in a non-phosphorylated state, increased their affinity for b-catenin dramatically. When phosphorylated, binding of the 20R to b-catenin was no longer modulated by the key aspartate in the Nterminal flanking region. Although the phosphorylation sites of Apc have not been identified in vivo, the fact that CK1 and GSK3b are endogenous components of the www.sciencedirect.com Regulation of cellular functions by the Apc protein McCartney and Näthke 187 Figure 1 Linear sequence schematic of Apc.The human Apc protein with 2843 amino acids is shown with the location of different functional sites. destruction complex suggests that the CK1 and GSK3b phosphorylation sites identified in vitro may be physiologically relevant [9]. These data significantly extend recently reported structural studies of the interaction between b-catenin and the 20R of Apc [10,11]. These studies demonstrated that when phosphorylated, 20R3 and its N-terminal and Cterminal flanking sequences make contact with the entire structural-binding groove of b-catenin. This contrasted significantly with binding of unphosphorylated 20R3 [10]. Phosphorylated Apc can compete with Axin for binding to b-catenin [10,12], but these proteins can bind simultaneously when Apc is not phosphoryated [10]. These observations suggest a model where phosphorylation of Apc is important for the recruitment and turnover of b-catenin in the destruction complex. Phosphorylation of Apc binds Axin and b-catenin simultaneously, helping to bring these components together (Figure 2). Once complexed, dephosphorylation of Apc reduces its affinity for b-catenin, and b-catenin is transferred to Axin, but retains a reduced interaction with Apc. Figure 2 Cycling of Apc phosphorylation may drive destruction complex function.(1) Apc with phosphorylated 20Rs has high affinity for b-catenin and may facilitate the entry of b-catenin into the complex (2). (3) Dephosphorylation of Apc, perhaps by PP2A, drives the hand-off of b-catenin to Axin where it can be phosphorylated by GSK3b. (4) P-b-catenin is identified by b-TrCP, the F-box subunit of the E3 ubiquitin ligase, ubiquitinated and degraded by the proteosome. Modified from [7]. www.sciencedirect.com Current Opinion in Cell Biology 2008, 20:186–193 188 Cell regulation b-catenin phosphorylated by Axin-associated GSK3b is targeted for ubiquitination and degradation by the proteosome. It is tempting to speculate that the phosphatase responsible for dephosphorylating Apc is PP2A. This phosphatase associates with Axin [13–15] and its regulatory subunit B56 interacts with both Axin and Apc through its Arm repeats [16,17]. Consistent with this model, mutations that disrupt the Arm repeats of Drosophila APC2 also disrupt the function of the destruction complex [18]. Ha et al. tested this model by exposing the phosphorylated-Apc/b-catenin complex to PP1, a biochemically tractable phosphatase similar to PP2A. Although PP1 could effectively dephosphorylate Apc alone, Apc complexed with b-catenin was resistant to its activity [10]. Although PP2A within the destruction complex may function differently, the mechanisms for dephosphorylating Apc within the complex clearly require more careful examination. Cycling of Apc phosphorylation has also been suggested to affect the release of b-catenin from the complex [12]. However, the presence of Apc in a complex with b-catenin and the ubiquitin ligase subunit b-TrCP [19] suggests that complete release from the destruction complex may not be necessary to hand over phosphorylated b-catenin to the proteosome [10] (Figure 2). Although many details of this pathway remain unknown, it is clear that phosphorylation of the 20R is a key step in regulating the binding of Apc to b-catenin. Furthermore, the different binding properties of each repeat suggest that they may play different roles in distinct cellular contexts [9]. In the unphosphorylated state, the binding affinities of any of the 20R except 20R3 are likely too low to be relevant. The different binding affinities in the phosphorylated state may be important to fine tune the Apc/b-catenin interactions that occur not only in the destruction complex, but also at other sites in the cell including the nucleus (see below), the cortex [20,21], and at the centrosome [22]. Further dissection of the repeats using a wide range of functional assays will be an essential step to understand this interaction. In the Wnt signalling context, identification of 20R3 as the highest affinity b-catenin binding repeat provides a more detailed explanation for the observation that the mutation cluster region (between amino acids 1250 and 1450 (Figure 1)) is associated with human colon cancer. Truncated proteins resulting from these mutations lack all of the Axin-binding SAMP repeats as well as 20R3–7, retaining only two of the weakest binding repeats 20R1-2. Nuclear Apc Phosphorylation of Apc is emerging as a key mechanism for regulating Apc function in the canonical destruction complex as well as in the regulation of Apc’s subcellular distribution. As part of its role as a negative regulator of Wnt signalling, Apc proteins shuttle in and out of the Current Opinion in Cell Biology 2008, 20:186–193 nucleus where they interact with b-catenin, CtBP, and other proteins involved in transcription (reviewed in [23]). Consistent with these observations, Apc contains two classic monopartite basic nuclear localisation signals in the C-terminal half of the protein and an additional domain within the Arm repeats that facilitates nuclear import (Figure 1). The regulation of Apc’s nuclear import is complex and involves a number of factors including the activity of several NESs (Figure 1), sequestration of Apc at non-nuclear sites by interactions with other protein partners, and regulation of NLS activity (reviewed in [23]). Regulation of NLS activity involves, at least in part, the phosphorylation of NLS flanking sites where phosphorylation by casein kinase 2 (CK2) may enhance NLS activity, while phosphorylation by protein kinase A (PKA) inhibits NLS activity [24]. Recent evidence suggests that p38 MAPK and its regulator Gadd45a act to enhance the nuclear import of Apc by enhancing the activity of CK2 and suppressing the activity of PKA [25]. The relationship between Apc and CK2 is complicated not only does CK2 appear to phosphorylate Apc but the direct binding of Apc to CK2 inhibits CK2 activity [26], suggesting that Apc imposes negative feedback on its own nuclear localisation. What is the function of nuclear Apc? Mounting evidence favours the hypothesis that Apc sequesters nuclear bcatenin. This is supported by careful observations of nucleo-cytoplasmic shuttling of b-catenin using live cell microscopy and fluorescence recovery after photobleaching (FRAP) [27], which revealed that none of the factors that bind b-catenin in the nucleus, including positive effectors TCF4, BCL9/Pygopus and negative regulators Apc and Axin influence the rate of b-catenin nuclear import and export. Rather, the data suggest that these proteins affect the compartmentalisation of b-catenin by sequestering it at specific sites. Taken together, these data support a model whereby p38 MAPK signalling activates CK2 to phosphorylate Apc, promoting its nuclear import where it acts to sequester b-catenin, perhaps in cooperation with CtBP [28], and so downregulates b-catenin activity. An interesting twist to this model was revealed by the finding that CK2 phosphorylates LEF1, and is required for the assembly and turnover of b-catenin/LEF1 nuclear complexes [29,30]. However, nuclear Apc may not be the only pool of Apc that regulates nuclear b-catenin. A recent study of C. elegans Apc, APR-1, suggests that cortical APR-1 functions to regulate the Wnt pathway primarily by facilitating the nuclear export of WRM1, a worm b-catenin [31]. Furthermore, although there has been significant progress in understanding the mechanisms that regulate nuclear Apc, its function in this compartment is not clear. There are many ideas that have not been explored in detail, particularly the possibilities that Apc itself may interact with DNA [32], and components of the base excision repair pathway [33]. www.sciencedirect.com Regulation of cellular functions by the Apc protein McCartney and Näthke 189 Figure 3 Apc in intestinal tissue.(a) In wild-type intestinal tissue, stem cells near the base of the crypt (red), give rise to transit amplifying cells (yellow) that differentiate into absorptive epithelial cells (green), neuroendocrine (brown), Paneth (blue), or Goblet (pink) cells. Differentiation is accompanied by active migration along the crypt-villus axis. Near the top of villi in the small intestine (or in the collar of the crypt in the colon, where villi are not present), cells are extruded and shed into the gut lumen while undergoing apoptosis (dark green). Apc contributes to this tissue organisation by supporting differentiation, thus keeping proliferation restricted to the appropriate compartment, and migration to ensure continuous efflux of cells from the proliferative compartment. It also contributes to apoptosis, and can enhance or inhibit cell death depending on the cellular context (position, tissue type etc.). (b) In the absence of Apc, or when only mutated Apc is present, cells do not differentiate normally; they remain proliferative and retain some stem cell characteristics (red-yellow). They also fail to migrate normally. Thus cells accumulate and form pre-malignant polyps that can deteriorate into adenoma and carcinoma. The top of villi retains normal enterocytes, because each villus is supplied with new cells by many crypts and usually only one crypt becomes malignant. Apc in apoptosis Mutations in Apc may also promote changes in apoptosis, a crucial component of epithelial biology in the intestinal tract. When cells reach the luminal collar of the colonic crypt or the tip of the intestinal villus they are extruded from the tissue while undergoing apoptosis and are shed into the lumen of the gut (Figure 3) [34]. Although the molecular mechanism for a role for Apc in apoptosis has not been identified, evidence for a role of Apc in the normal execution of an apoptotic program is accumulating (reviewed in [35]). Whether components of the cell death pathway are direct targets of Wnt signalling has yet to be elucidated, though screens for Wnt target genes have detected changes in genes encoding proteins directly involved in the execution of apoptosis [36,37]. However, other changes induced by Apc mutations (see below) may also contribute [38]. Importantly, the overall balance of pro-apoptotic and anti-apoptotic changes induced by Apc loss may be highly context- and tissue-specific and may cause increased or decreased apoptosis, depending on tissue type, tissue compartment, developmental stages, etc. www.sciencedirect.com Apc as a cytoskeletal regulator important in migration, orientation and division The transcriptional changes induced by mutant Apc provide the background against which the other cellular functions of Apc become particularly important in the digestive tract epithelium. In addition to its function in the b-catenin destruction complex, Apc can also directly contribute to the regulation of cytoskeletal proteins: it binds directly and indirectly to microtubules and also interacts with a number of actin-regulatory proteins (Figures 1 and 4). So it is not surprising that loss of Apc leads to changes in cell migration, cell orientation, polarity and division [38,39,40,41]. Transcriptional changes induced by Apc mutations also contribute to these defects; nonetheless, direct interactions of Apc with cytoskeletal elements contribute significantly and directly. The stabilising effect on microtubules by Apc has been established biochemically [42,43] and in cells [39,44] and may be affected by the interaction of Apc with the microtubule plus tip-binding protein EB1 [45]. The ability of C-terminal fragments of Apc to nucleate the assembly Current Opinion in Cell Biology 2008, 20:186–193 190 Cell regulation Figure 4 Figure 5 Apc complexes in migrating cellular protrusions. A number of proteins support the linkage between Apc and microtubules, but also F-actin in migrating cellular protrusions near the plasma membrane. Whether all of these interactions can occur at the same time, and how they affect each other is not known. Apc in mitotic spindles.Apc (red dot) localises to spindle microtubules, concentrates at kinetochores, centrosomes and at sites of cortical attachment. At these different sites Apc may co-operate with the indicated proteins to support microtubule attachment and dynamics at both kinetochores and cortical sites, but also at centrosomes. of F-actin in vitro [46] together with the ability of Apc to interact with a number of actin-regulatory proteins including ASEF, an exchange factor for CDC42 and Rac, and IQGAP, suggests that Apc also contributes to the regulation of the cellular F-actin network [47,48]. The idea that Apc helps to co-ordinate the F-actin and microtubule cytoskeletons is further supported by the interaction of Apc with mDia, a protein that can affect both actin and microtubules [49] (Figure 4). How these interactions are co-ordinated, whether they occur simultaneously or whether these proteins compete with each other, and how they contribute individually or together to the localised recruitment of Apc clusters in migrating cellular protrusion is not at all clear and will require careful biochemical and functional experiments (Figure 4). function of the microtubule-based mitotic spindle (Figure 5). Loss of Apc leads to defects in spindle alignment [51], and the mitotic spindle checkpoint [38], and to the development of anaphase bridges [52]. An immediate consequence of these changes is the accumulation of tetraploid cells, not only in culture, but also in intestinal tissue depleted of Apc [38]. Similar defects are observed in cells expressing N-terminal fragments of Apc [53] and intestinal tissue from Min mice displays failures in cytokinesis [54]. The expression of Nterminal fragments of Apc, similar to those present in human tumours, may disrupt normal Apc function by forming complexes with wild-type Apc and interfering with its normal interactions [55]. One possible interaction that may be affected by such Apc heterodimeric complexes is the binding of Apc to EB1 [56]. Nonetheless, it has been clearly established that these interactions contribute to a number of cellular processes that are crucial for gut epithelium maintenance, most prominently in cell migration. Differentiating epithelial cells in the gut actively migrate upwards from the crypts and loss of Apc leads to a decrease in this migration [50] (Figure 3). Similarly, depleting Apc from cultured cells leads to a decrease in the ability to migrate in a directed fashion [39] that is accompanied by a decrease in overall microtubule stability and loss of polarisation of acetylated microtubules towards the leading edge [39,44]. Similar to migrating epithelial cells, highly polarised migrating astrocytes also accumulate Apc at their leading edge and loss of Apc causes a loss of migration in these cells [40]. The cytoskeletal interactions of Apc not only affect cells in interphase but also contribute significantly to the Current Opinion in Cell Biology 2008, 20:186–193 The interaction between Apc and EB1 and thus dynein/ dynactin, which binds to EB1 directly, may also contribute to the ability of Apc to support anchoring of astral microtubules at cortical sites (Figure 5) [54,57–59]. Similarly this interaction may contribute to the function(s) of Apc at the centrosome [22]. Together these functions of Apc in the mitotic spindle could explain the spindle positions defects in Apc mutant cells [53]. Apc in whole tissue Much of the evidence for the functions of Apc is based on data obtained in cultured cells. However, important insights into how these functions contribute to tissue homeostasis and tumourigenesis have been gained using animal models. Mice with constitutive or conditional mutations in Apc confirmed its contribution to differenwww.sciencedirect.com Regulation of cellular functions by the Apc protein McCartney and Näthke 191 tiation, migration, division and genetic stability: both Min mice and PIRC rats are heterozygous for Apc mutations, similar to FAP patients, and both develop tumours in the digestive tract similar to those in human FAP patients [60]. Conditional depletion of Apc specifically in the intestine results in major changes in cell differentiation, proliferation, ploidy and apoptosis consistent with the findings in cell culture systems [50]. Importantly, the defects induced by acute loss of Apc in gut tissue do not occur when c-myc is also missing [61]. Cells lacking both of these genes are selectively depleted from intestinal tissue within a few weeks. It is possible that a downstream target of c-myc that is upregulated when Apc is lost is responsible for the migration defect observed in Apcdepleted cells. One difficulty in measuring a change in migration in c-myc-depleted cells in this context is that these cells do not survive and are out-competed rapidly by wild-type cells in this tissue. This means that mixed populations of cells, plus and minus c-myc, co-exist and it is impossible to resolve how mixing between such cells affects migration and general cell behaviour. However, during the lifetime of these double-mutant cells in intestinal tissue, b-catenin is nuclear suggesting that it is acting as a transcriptional activator [61]. These data suggest that upregulation of c-myc is absolutely required for Apc-deficient cells to survive. Conclusion Many of the defects that result from Apc-deficiency can be overcome by overexpressing other components of the relevant pathways: overexpression of Axin can rescue b-catenin regulation in Apc-deficient cells [62], cell cycle checkpoint deficiency can be rescued by overexpressing Bub1 [38]. Furthermore, Apc is not absolutely required for any of the processes discussed above and most of them continue to proceed, albeit less efficiently in its absence: cells still move, but more slowly and not as directed; cells divide, but make more mistakes in this process; apoptotic pathways still function, but are not initiated appropriately in response to certain checkpoints [38]. In support of the notion that Apc is not essential for these processes, Drosophila completely lacking Apc do not exhibit detectable defects in mitosis, cytokinesis, cell polarity, or adhesion, but lethality appears to result primarily from excessive Wnt signalling [18]. A role for Apc in ensuring accuracy and efficiency of many cellular processes explains why Apc mutations are particularly detrimental for tissues that rely on the fidelity of these processes. The cumulative destabilisation that results from Apc disruption makes Apc a perfect tumour suppressor, particularly in gut epithelial tissues: loss of Apc leads to a large increase in mistakes in the processes that govern the overall homeostasis of this tissue and these mistakes are not corrected, instead they are tolerated allowing these oncogenic changes to propagate. www.sciencedirect.com References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser A, Luongo C, Gould KA, Dove WF: Multiple intestinal neoplasia caused by mutations in the murine homolog of the APC gene. Science 1992, 256:668-670. This paper identifies the Apc locus as the site for mutations in intestinal neplasia. 2. Moser AR, Dove WF, Roth KA, Gordon JI: The Min (multiple intestinal neoplasia) mutation: its effect on gut epithelial cell differentiation and interaction with a modifier system. J Cell Biol 1992, 116:1517-1526. This paper identifies the Apc locus as the site for mutations in intesitnal neplasia, but also demonstrates the effects of genetic background on penetrance of phenotype 3. Moser AR, Luongo C, Gould KA, McNeley MK, Shoemaker AR, Dove WF: ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur J Cancer 1995, 31A:1061-1064. This paper established the causal relationship between Apc heterozygosity and intestinal neoplasia. The Min mouse described in this paper is one of the most commonly used mouse strains in the world. 4. Rubinfeld B, Souza B, Albert I, Müller O, Chamberlain SH, Masiarz FR, Munemitsu S, Polakis P: Association of the APC gene product with b-catenin. Science 1993, 262:1731-1734. This paper shows the interaction of Apc protein with b-catenin, which set the stage for the identifying the Wnt-regulated degradation of b-catenin as a central pathway in colon tumour development. 5. Su L-K, Vogelstein B, Kinzler KW: Association of the APC tumor suppressor protein with catenins. Science 1993, 262:17341737. Shows the interaction of Apc protein with b-catenin, which set the stage for the identifying the Wnt-regulated degradation of b-catenin as a central pathway in colon tumour development. 6. Polakis P: The many ways of Wnt in cancer. Curr Opin Genet Dev 2007, 17:45-51. 7. Kennell J, Cadigan K: APC and ß-catenin degradation. In APC proteins. Edited by Näthke IS, McCartney BM: Landes Bioscience; 2008, in press. http://www.eurekah.com/chapter/3776. 8. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P: Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 1996, 272:1023-1026. This paper provides significant insights into the assembly and regulation of the complex that governs the degradation of b-catenin. Of particular importance to this discussion was the finding that the phosphorylation of the central repeat region of Apc significantly enhances its affinity for bcatenin. 9. Liu J, Xing Y, Hinds TR, Zheng J, Xu W: The third 20 amino acid repeat is the tightest binding site of APC for beta-catenin. J Mol Biol 2006, 360:133-144. The authors present a detailed analysis of the binding affinities of individual phosphorylated and unphosphorylated 15R and 20R for bcatenin. They showed that individual repeats have distinct binding affinities, with 20R3 being the highest, and the phosphorylation increases the binding affinities for all of them. Furthermore, specific amino acids in the 20R N-terminal flanking sequences significantly enhance the binding affinities of unphosphorylated 20R for b-catenin. Further study is required to determine the functional significance of these distinctions. 10. Ha NC, Tonozuka T, Stamos JL, Choi HJ, Weis WI: Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. Mol Cell 2004, 15:511521. See annotation to [11]. 11. Xing Y, Clements WK, Le Trong I, Hinds TR, Stenkamp R, Kimelman D, Xu W: Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function. Mol Cell 2004, 15:523-533. This paper along with [10] reported the crystal structure of Apc 20R complexed with b-catenin. These studies made the surprising observaCurrent Opinion in Cell Biology 2008, 20:186–193 192 Cell regulation tion that when phosphorylated not only does the 20R itself interact with bcatenin, but N-terminal and C-terminal flanking regions make contact as well. Ha et al. [10] demonstrated that phosphorylated Apc (P-Apc) competes with Axin for binding to b-catenin, while non-P-Apc and Axin can bind b-catenin simultaneously. These findings suggest a novel model for interactions within the destruction complex. Phosphorylation may be a ‘critical switch’ for Apc outside of the destruction complex as well. Xing et al. 2004 showed that P-Apc competes with Tcf binding to b-catenin, and thus may provide an additional mechanism for the downregulation of b-catenin transcription by Apc. 12. Xing Y, Clements WK, Kimelman D, Xu W: Crystal structure of a beta-catenin/axin complex suggests a mechanism for the betacatenin destruction complex. Genes Dev 2003, 17:2753-2764. This paper provided important structural insights into the nature of the bcatenin-Axin complex that set the stage for the later structural work. 13. Gao ZH, Seeling JM, Hill V, Yochum A, Virshup DM: Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci U S A 2002, 99:1182-1187. 14. Ikeda S, Kishida M, Matsuura Y, Usui H, Kikuchi A: GSK-3betadependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. Oncogene 2000, 19:537545. 15. Hsu W, Zeng L, Constantini F: Identification of a domain of Axin that binds to the serin/threonine protein phosphatase 2A and a self-binding domain. J Biol Chem 1999, 274:3439-3445. 16. Seeling J, Miller J, Gil R, Moon R, White R, Virshup D: Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 1999, 283:2089-2091. This paper demonstrates the involvement of PP2A as an important regulator of b-catenin degradation. 17. Yamamoto H, Hinoi T, Michiue T, Fukui A, Usui H, Janssens V, Van Hoof C, Goris J, Asashima M, Kikuchi A: Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A. J Biol Chem 2001, 276:26875-26882. 18. McCartney BM, Price MH, Webb RL, Hayden MA, Holot LM, Zhou M, Bejsovec A, Peifer M: Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila. Development 2006, 133:2407-2418. The authors showed that Drosophila null for both Apc genes (Apc1 and APC2) do not exhibit significant defects in a variety of processes in which Apc has been implicated, including cell viability, cadherin-based adhesion, spindle morphology and orientation, or cytokinesis. Complete loss of Drosophila Apc proteins does result in significant activation of Wnt signalling throughout development. These findings are consistent with the hypothesis that although Apc proteins may function in many cellular processes, they are not strictly required for most of these processes, but rather function in their fidelity and/or efficiency. 19. 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Edited by Näthke IS, McCartney BM: Landes Bioscience; 2008, in press. http:// www.eurekah.com/chapter/3835. 24. Zhang F, White RL, Neufeld KL: Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein. Proc Natl Acad Sci U S A 2000, 97:1257712582. Current Opinion in Cell Biology 2008, 20:186–193 This paper provided key evidence that the NLS activity of Apc is regulated by the phosphorylation of NLS flanking sites in two different ways. Phosphorylation by casein kinase 2 appears to enhance NLS activity, while phosphorylation by protein kinase A, inhibits NLS activity. 25. Hildesheim J, Salvador JM, Hollander MC, Fornace AJ Jr: Casein kinase 2- and protein kinase A-regulated adenomatous polyposis coli and beta-catenin cellular localization is dependent on p38 MAPK. J Biol Chem 2005, 280:17221-17226. 26. Homma MK, Li D, Krebs EG, Yuasa Y, Homma Y: Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein. Proc Natl Acad Sci U S A 2002, 99:59595964. 27. Krieghoff E, Behrens J, Mayr B: Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention. J Cell Sci 2006, 119:1453-1463. Using live cell imaging and fluorescence recovery after photobleaching, the authors demonstrated that none of the factors known to bind to bcatenin in the nucleus influence the rate of b-catenin nuclear import and export. Instead, these binding partners may act to sequester b-catenin in specific cellular compartments. This elegant study strongly supports the model that one function of Apc in the nucleus is to sequester b-catenin and thus downregulate its transcriptional activity. 28. Hamada F, Bienz M: The APC tumor suppressor binds to Cterminal binding protein to divert nuclear beta-catenin from TCF. Dev Cell 2004, 7:677-685. 29. Wang S, Jones KA: CK2 controls the recruitment of Wnt regulators to target genes in vivo. Curr Biol 2006, 16:2239-2244. See annotation to [30]. 30. Tapia JC, Torres VA, Rodriguez DA, Leyton L, Quest AF: Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factordependent transcription. Proc Natl Acad Sci U S A 2006, 103:15079-15084. This paper along with [29] provide evidence for additional roles for casein kinase 2 (CK2) in Wnt signalling. Not only does CK2 appear to regulate the nuclear localisation of Apc (24), but it also plays a role in the assembly of LEF1-b-catenin transcriptional complexes. 31. Mizumoto K, Sawa H: Cortical beta-catenin and APC regulate asymmetric nuclear beta-catenin localization during asymmetric cell division in C. elegans. Dev Cell 2007, 12:287299. 32. 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Kroboth K, Newton IP, Kita K, Dikovskaya D, Zumbrunn J, Waterman-Storer CM, Näthke IS: Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability. Mol Biol Cell 2007, 18:910-918. This paper shows that Apc status correlates with over microtubule stability in cells and affects cell migration. 40. Etienne-Manneville S, Hall A: Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 2003, 421:753-756. This paper provides important link between small GTPases and Apc in migrating protrusions. 41. Etienne-Manneville S, Manneville JB, Nicholls S, Ferenczi MA, Hall A: Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization. J Cell Biol 2005, 170:895-901. 42. Munemitsu S, Souza B, Müller O, Albert I, Rubinfeld B, Polakis P: The APC gene product associates with microtubules in vivo and promotes their assembly in vitro. Cancer Res 1994, 54:3676-3681. This is the first demonstration that Apc can bind to microtubules. 43. Zumbrunn J, Inoshita K, Hyman AA, Näthke IS: Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3b phosphorylation. Curr Biol 2001, 11:44-49. This paper shows the direct effect of Apc on microtubule stability in vitro and in cells and provides evidence that this function can be regulated by the phosphorylation with GSK3b. 44. Kita K, Wittmann T, Näthke IS, Waterman-Storer CM: APC on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1. Mol Biol Cell 2006, 17:2331-2345. This paper demonstrates direct link between endogenous Apc protein and microtubule dynamics in cells. 45. Su L-K, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, Trent J, Vogelstein B, Kinzler KW: APC binds to the novel protein EB1. Cancer Res 1995, 55:2972-2977. This paper identifies EB1 as an Apc binding partner and thus brings Apc closer to the microtubule cytoskeleton. 46. Moseley JB, Bartolini F, Okada K, Wen Y, Gundersen GG, Goode BL: Regulated binding of adenomatous polyposis coli protein to actin. J Biol Chem 2007, 282:12661-12668. 47. Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T: Asef, a link between the tumor suppressor APC and G-protein signaling. Science 2000, 289:1194-1197. Identifies Apc as a regulator for the activity of an exchange factor for small GTP-ases thus establishing a functional link between Apc and actin. 48. Watanabe T, Wang S, Noritake J, Sato K, Fukata M, Takefuji M, Nakagawa M, Izumi N, Akiyama T, Kaibuchi K: Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev Cell 2004, 7:871-883. 49. Wen Y, Eng CH, Schmoranzer J, Cabrera-Poch N, Morris EJ, Chen M, Wallar BJ, Alberts AS, Gundersen GG: EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat Cell Biol 2004, 6:820-830. 50. Sansom OJ, Reed KR, Hayes AJ, Ireland H, Brinkmann H, Newton IP, Batlle E, Simon-Assmann P, Clevers H, Näthke IS et al.: Loss of Apc in vivo immediately perturbs Wnt www.sciencedirect.com signaling, differentiation, and migration. Genes Dev 2004, 18:1385-1390. Shows that conditionally inactivating Apc in the gut immediately leads to changes that underlie tumourigenesis. 51. Draviam VM, Shapiro I, Aldridge B, Sorger PK: Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells. EMBO J 2006, 25:2814-2827. 52. 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Green RA, Wollman R, Kaplan KB: APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Mol Biol Cell 2005, 16:4609-4622. 57. Berrueta L, Tirnauer JS, Schuyler SC, Pellman D, Bierer BE: The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain. Curr Biol 1999, 9:425-428. 58. Strickland LI, Wen Y, Gundersen GG, Burgess DR: Interaction between EB1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis. Curr Biol 2005, 15:2249-2255. 59. McCartney BM, McEwen DG, Grevengoed E, Maddox P, Bejsovec A, Peifer M: Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin. Nat Cell Biol 2001, 3:933-938. 60. Amos-Landgraf JM, Kwong LN, Kendziorski CM, Reichelderfer M, Torrealba J, Weichert J, Haag JD, Chen KS, Waller JL, Gould MN et al.: A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer. Proc Natl Acad Sci U S A 2007, 104:4036-4041. This paper introduces a novel rat model for Apc-mediated tumourigenesis that mimics the human disease more accurately. 61. Sansom OJ, Meniel VS, Muncan V, Phesse TJ, Wilkins JA, Reed KR, Vass JK, Athineos D, Clevers H, Clarke AR: Myc deletion rescues Apc deficiency in the small intestine. Nature 2007, 446:676-679. This paper demonstrates that Myc is absolutely required for Apc-deficient cells to survive in intestinal tissue and form tumours and that de-regulated, nuclear b-catenin is not sufficient. 62. Hart MJ, de los Santos R, Albert I, Rubinfeld B, Polakis P: Downregulation of ß-catenin by human axin and its association with the APC tumor suppressor, ß-catenin and GSK3ß. Curr Biol 1998, 8:573-581. 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